A method for the determination of chlorophylls a and b, carotene, and total xanthophyll from a single ether solution is described. This method is adaptable to any spectrophotometer of good resolving power provided preparations are available for determination of absorption constants for the instrument to be used. The pigments are extracted from the plant material with acetone, transferred to ether, and chlorophyll is determined from the light absorption at Chlorophyll Mo./g. Mo./o. Mo./o. Mg./sq. m. Mg./sq.m. MoJtq. m.

N A number of the methods for determining chlorophyll and the carotenoids in plants the sample is extracted with acetone and the pigments are transferred to ether by washing with water a number of times in separatory funnels {1-4). This procedure was followed in the earlier work in this laboratory (S).The time required for these analyses can be decreased considerably by means of the apparatus herein described. The pigments are transferred to ether by passing an acetone extractether mixture through a vertical column in which water is moving in the direction opposite to the flow of the mixture. As the mixture bubbles through the water, the acetone is removed by the water and the ether solution collects at the top of the column.By changing the water level in the column, the washed ether solution is driven into a cooling coil which decreases the solubility of water in ether and dries the solution sufficiently for immediate pigment determinations.

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In conlnection with the tobacco breeding program at the Kentucky Agricultural Experiment Station, a large number of varieties of tobacco are available, of which a part of the genetic constitution is known. An investigation was made of the chlorophyll a and b and carotene contents of eighteen of these varieties.The method of inheritance of the white burley character has been studied by HENIKA (2). He found that duplicate recessive genes were responsible for this lighter colored type of tobacco but did niot conduct any chemical analyses to show the difference between types. Our purpose was to find out the differences in pigment content of the burley and dark tobaccos and the difference, if any, in the pigment content of mosaic-susceptible and mosaicresistant varieties.Methods MATERIAL USED Seed from 7 dark and 11 burley tobacco varieties were planted on March 2, 1942. The seedlings were all transplanted from the seed bed to individual pots and from the pots to a ground bench in the greenhouse. The plants were grown under similar environmental conditions and were in bloom but had not been topped when the samples were taken, July 13 to 31, 1942. Duplicate samples of two leaves each were taken from a plant. The four leaves came from the lower half of the plant at a height of approximately 1.5 ft. from the ground and were consecutive leaves on the stalk. The length and width of the leaves were measured to the nearest cenitimeter and then the midribs were removed. The right half of one leaf and the left half of the other leaf of each sample were analyzed; the other half of each leaf was used for moisture percentage determination. The moisture sample was dried in an oven at 650 C. for 48 hours or longer. The percentag,e dry weight of the sample analyzed was assumed to be the same as that of the moisture sample.YOUNG and JEFFREY (4) found that there was a high degree of correlation between the product of length and width and the leaf area. Their equation was used in determining leaf area. Since the equation used was developed for Ky. 16, the calculated leaf area of those varieties which have a different shape of leaf may be inaccurate. This is particularly true of the species Nicotiana digltta (F3 Ky. 5 x N. gluttinosa) which has a petiolate leaf. The calculated area of leaves of this strain is probably too great, resulting in chlorophyll and carotene values which are too low when expressed on an area basis.

IntroductionChanges in the chemical composition of burley tobacco which take place in curing are being investigated at the Kentucky Agricultural Experiment Station in an attempt to understand the physiology of curing and its effect on leaf quality. Color is recognized as one of the most important factors in the determination of leaf quality, any retention of green pigment in the cured leaf greatly reducing its value. The chlorophylls are the green pigments in normal tobacco, and the investigations reported in this paper were made primarily to determine the rate and amount of change in these pigments during air curing on the stalk of tobacco cut at three stages of maturity. The development of a method for determining carotene from the same solution used in the chlorophyll determinations made possible the study of this constituent.This study of the catabolic changes of plant pigments is of general biochemical and physiological interest. Numerous articles have appeared reporting the rates of formation of these pigments under different conditions, but studies on pigment disappearance are rare. In 1918 WILLSTXTTER and STOLL (5) reported that the chlorophyll content of yellowed leaves was less than of green leaves. They did not, however, follow closely the loss of either chlorophyll or carotenoids. GUTHRIE (2) investigated the pigment changes in potted plants when placed in the dark as compared with others remaining in the light. His results indicated that tomato plants lost about 25% of their chlorophyll in four days, soybean plants about 70%o in eight days, and yellow coleus practically none in eight days.

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ALFALFA has long been of prime importance as a hay and forage crop because of its high protein and carotene contents. Protein is generally stable during storage, but may be lost in leaf shatter through poor management in handling the crop. Carotene, on the other hand, is very unstable and losses may be severe during harvest operations and storage. In spite of customary large losses of carotene, alfalfa products are of great importance in the feeding of livestock and poultry because of their content of beta-carotene (provitamin A). This is largely due to the initial high content of this nutrient rather than to superior methods of preventing loss. Ham and Tysdal (1946) stated that .certain crosses may be consistently different in carotene content from others. Thompson (1949) has recently discussed the desirability of obtaining alfalfa varieties with higher carotene content and indicated that differences exist among common varieties. This study was undertaken to fill a need for further and more comprehensive survey work on contents of both carotene' and protein. While extensive comparisons were being made of the carotene contents of different varieties, hybrids, and selections from the alfalfa-breeding project at this station, several factors influencing the results of carotene determinations were evaluated. A rapid and satisfactory method of sampling and comparing the carotene contents of alfalfa varieties was developed. Consideration of these factors may be applicable to studies of other constituents in alfalfa and related crops. MATERIALS AND METHODS The alfalfa was grown on the University Experiment Station Farm at Davis, California, in a field of uniform Yolo fine sandy loam. The alfalfa was planted in close-drilled rows (6 inches apart), in plots 3% x 16 feet in